Heart from aging animal is more susceptible to ischemic injury. Mitochondria isolated from aging rats have defects in complex III of electron transport chain. In addition, complex III is also the main site of defect in mitochondria isolated from young and old rats after ischemia. Recently, we observed that cell-permeable ceramides also inhibit mitochondria isolated from rat liver, heart, and muscle at the complex III site of electron transport chain. A number of studies have shown that ceramide-related lipids are elevated in aging cells or animals. In addition, tumor necrosis factor alpha (TNF), which increase cell ceramide concentration by activating sphingomyelinase, is elevated in ischemic tissues and in aging animals. Besides aging and ischemia, a number of other metabolic conditions characterized by abnormal fatty acid metabolism are also associated with elevated TNF expression. Furthermore, it is known that increased utilization of fatty acids by hearts exaggerates the injury of hearts after ischemia-reperfusion. It appears that there is a causal relationship between abnormal fatty acid metabolism and the onset of tissue damage which might be mediated through abnormal cytokine expression and sphingolipid signal transduction. The hypothesis to be tested is that elevated tissue ceramide and TNF content, which lead to a depressed mitochondrial complex III, in aging and cardiac ischemia contribute to the increased susceptibility of aging hearts to ischemic injury. Complex III is one of the major sites of oxygen free radical production. A inhibition of complex III increased the production of free radical which leads to tissue damaged and depressed heart functions. In this proposal, we will characterize cardiac cytokine expression and lipid metabolism in relation to aging abnormality in cardiac mitochondria and function using isolated buffer perfused Fischer 344 rat hearts as model. Rats at 6, 18, 24, and 28 month age will be studied. Furthermore, the abnormality in aging hearts can be duplicated by elevated fatty acid content in perfusion medium and by inhibiting fatty acid oxidation in young rats. Interventions aimed at preventing fatty acid flux into cardiac tissue, the accumulation of TNF, and the production of ceramides should protect aging hearts from exaggerated ischemia- reperfusion injury. These interventions could include carnitine palmitoyltransferase-I inhibitors, anti-TNF antibodies, and inhibitors which block the production of ceramides from sphingomyelin hydrolysis of sphingosine acylation.